Project 1 is investigating the mechanismof electrical rhythmicityin GImuscles. Phasiccontractions are timed by electrical slow waves that are generated by a specializedpopulationof cells known as interstitial cells of Cajal (ICC). We developed a cell culture model of ICCduringthe previous funding period. These cells are spontaneously active, producingspontaneous transient inward currents (STICs), but the cells do not fully recapitulate electrical rhythmicityin intact musclestrips. For example,we could not activate large amplitude currents responsible for slow waves in these cells. Molecular studies showed that cultured ICC rapidly dedifferentiate and lose the ICC phenotype withina few days in culture. Afresh preparation of ICCwas difficult to develop because ICC are difficult to identify in mixedpopulationsof cells. In the next funding period we will utilize a new genetic tool we have created by engineeringexpression of a bright green fluoresccent protein (copGFP) in ICC. Expression of the reporter makes it easy to find ICCin cell dispersions and to sort ICCby fluorescence activated cell sorting for molecularstudies. Cells from these mice displayspontaneous rhythmicity in the form oflarge amplitude spontaneous depolarizations that we believe are equivalent to slow waves in intact ICC networks. We will characterize the spontaneous activityof single ICCand describe the properties of the large amplitude 'autonomous'currents that underlyspontaneous depolarization. Autonomouscurrents can be pace by depolarization of single cells. We will explore the voltage-dependence of autonomous currents and determine the underlyingvoltage-sensor and mechanismthat initiates these currents. We will also investigate how spontaneous activity in single ICCis regulated with the goal of understanding how slow wavefrequency (and ultimately phasic contractile activity) is regulated. Several clinical studies have demosntrate loss of ICCin pathophysiological conditions, and this has resulted in hypotheses about the cause of GI motilitydisorders. We hypothesize that changes in ICCmay precede the loss of cells, and we will characterize changes in ICCfunction during the development of type II diabetes. We will also study changes in ICCduring the development of hyperplasia in pre-GIST ICCnetworks. New ideas about pacemaker activity in the gut will result from this work. RELEVANCE (See instructions): Interstitial cells of Cajal generate pacemaker activity in the GI tract that is responsible for peristaltic and segmental contractions in motility. Defects in pacemaker activity lead to disordered motility and unregulated transit of food and nutrients. Understanding how pacemaker cells work will provide new ideas about how to control GI motility and offer new suggestions about how to treat GI motility disoders.